Bladder wall abnormalities in myelodysplastic bladders: a computer assisted morphometric analysis

Myelodysplasia represents the most common cause of neurogenic bladder dysfunction in children. The specific histological features associated with myelodysplastic bladders have not been previously characterized. Our objective was to study the relationship between smooth muscle and connective tissue in control and myelodysplastic bladders using classical morphometric analysis with the assistance of an automated image analysis system. Gross histological analysis of the bladder specimens of normal stillborn fetuses showed organized muscle bundles embedded in a small amount of connective tissue. The bladder specimens of myelomeningocele stillborn fetuses showed a marked paucity of muscle bundles as well as a significantly diminished size of the muscle bundles. The myelomeningocele bladder specimens obtained from patients undergoing autopsy and those undergoing augmentation cystoplasty revealed significant interfascicular and pericellular infiltration of the smooth muscle by dense connective tissue. Quantitative morphometric analysis showed that the myelomeningocele stillborn fetuses have a significant increase in the volumetric content of connective tissue compared to control stillborn fetuses. The bladders of myelomeningocele patients who underwent autopsy or augmentation cystoplasty had a 3-fold increase in connective tissue when compared to normal controls. These findings reveal that structural changes in the histological components of the myelodysplastic bladder can be demonstrated not only in patients of varying ages undergoing autopsy or augmentation cystoplasty but also in the developing fetus. These findings enhance our understanding of the relationship of connective tissue proliferation to smooth muscle in the myelodysplastic bladder. We discuss the relationship of these findings to pathological detrusor morphology and detrusor dysfunction.     

Structural changes in the bladder walls of pregnant and hormone-treated rats: correlation with bladder dynamics

OBJECTIVE: To evaluate the effects of oestrogen, progesterone and pregnancy on bladder dynamics, and assess the associated histological and structural changes in the bladder wall in a rat model. MATERIALS AND METHODS: Adult female Sprague-Dawley rats were divided into groups and treated as follows: nonpregnant controls (NC); pregnant (PG); oophorectomized and treated with oestrogen (ES); oophorectomized and treated with progesterone (PR); and oophorectomized controls (OO). Simultaneous and continuous renal pelvic and bladder pressures were recorded during bladder filling and emptying. Connective tissue and smooth muscle were analysed morphometrically and quantitatively, and immunohistochemistry used to evaluate the distribution and expression of collagen types I, III and IV. RESULTS: PG rats had significantly greater bladder compliance than NC, OO and PR rats (P < 0.05). This correlated with the morphometric analysis, with bladders of PG and PR rats having lower connective tissue to smooth muscle ratios than NC, ES and OO rats (P < 0.05). Collagen I was increased in the lamina propria of PG and ES rats, and the detrusor muscle layer showed greater localization of collagen III in the interfascicular space in the PG and PR rats than in the other groups. There was no change in the distribution of collagen IV. CONCLUSION: PG and PR rats had the highest bladder compliance and the changes paralleled structural changes in the bladders, specifically in the ratio of connective tissue to smooth muscle, and the distribution and expression of collagens I and III. These changes have age-related implications in the urinary tract.     

Histamine content and mast cell count of detrusor muscle in patients with interstitial cystitis and other types of chronic cystitis

The quantitative mast cell count in the detrusor muscle, the histamine content and the degree of collagen staining material in the bladder wall have been evaluated in order to elucidate their value in distinguishing between patients with interstitial cystitis and other types of chronic cystitis. The number of mast cells in the detrusor muscle was statistically significantly increased in patients with interstitial cystitis compared with the control group (P less than 0.0001). With a proposed level of greater than 20 mast cells/sq mm of muscle tissue the diagnostic specificity was 88% and the diagnostic sensitivity 95%. The histamine content in the bladder wall was significantly increased in patients with interstitial cystitis (P less than 0.05) but not useful as a diagnostic test. The amount of collagen staining material was significantly increased in the intra- and inter-fascicular muscle tissue of the bladder in patients with interstitial cystitis (P less than 0.0005, P less than 0.001) and might be used as a support for the histological diagnosis, even in patients with uncontracted bladders.     

Structure of trabeculated detrusor smooth muscle in cases of prostatic hypertrophy

The light and electron microscopic structure of biopsy samples of trabeculated urinary bladder from patients with proven outlfow obstruction due to prostic hypertrophy has been compared with the morphology of control bladder specimens. In the latter the detrusor muscle bundles were composed of smooth muscle cells closely packed together with very little intervening connective tissue. In contrast, irrespective of age, detrusor muscle from trabeculated bladders contained many muscle bundles in which the constituent cells were of relatively small diameter and were widely separated from each other by dense masses of connective tissue. No morphological evidence for smooth muscle hypertrophy or hyperplasia was obtained in the present study. In the electron microscope the connective tissue between the smooth muscle cells of trabeculated bladders was seen to contain, in addition to collagen fibrils, an extensive meshwork of electron-dense microfibrils apparently in continuity with the basal laminae of the smooth muscle cells. Regions of close approach between smooth muscle cells were seemingly unaffected by trabeculation as was the distrubution and fine structure of autonomic nerve terminals. These observations are intended to form a baseline for comparision with the results of future morphological studies of trabeculation arising in response to different aetiological factors.     

Focal changes in nerve, muscle and connective tissue in normal and unstable human bladder

OBJECTIVE: To compare and quantify, in a morphological study, the changes that occur in the connective tissue elements (elastin and collagen), muscle fibre diameters and nerve densities between normal, idiopathic and neuropathic bladders. MATERIALS AND METHODS: Bladder tissue was obtained from 27 patients undergoing cystectomy for carcinoma, from 12 with idiopathic instability and from seven neuropathic patients who were undergoing ileocystoplasty. A combination of histochemical and immunohistochemical techniques were used to detect detrusor muscle, connective tissue and nerve profiles in the bladder tissue. RESULTS: In both idiopathic and neuropathic bladder tissue the structural changes were highly punctate. From the density of nerve profiles, three areas were defined: (i) apparently unaffected normal fascicles with a high density of nerves, no hypertrophy of the muscle and no infiltration of elastin and collagen. The nerve density in these areas was similar to that in normal bladder tissue. (ii) Fascicles with a low density of nerve profiles, muscle hypertrophy but no connective tissue infiltration. (iii) Areas with few nerve profiles, muscle hypertrophy and extensive elastin and collagen infiltration within the fascicles. The mean (sem) density of nerve profiles in control tissue was 752 (53) nerves/mm2 and in the idiopathic bladders was 905 (91), 81 (20) and 74 (38) nerves/mm2 in the three defined areas, respectively. In the neuropathic tissues the nerve profile densities were 672 (249), 57 (23) and 37 (28) nerves/mm2, respectively. Fibre diameter, elastin and collagen content and nerve density were measured in normal and unstable bladder tissue using these three defined areas. The mean (sem) fibre diameter was 6.81 (0.52) in normal bladder; in idiopathic bladder tissue the fibre diameters in the three areas were 6.72 (0.62), 7.06 (0.62) and 7.34 (1.15) micrometer, respectively, and in neuropathic bladders were 6.75 (0.62), 8.24 (0.62) and 9.35 (0.62) micrometer, respectively. The relative areas of elastin were 0.79 (0.70), 0.56 (0.45) and 18.3 (4.1)% for the control, normal and affected areas of the neuropathic bladders, respectively, and the relative areas of collagen were 3.5 (1.3), 6.15 (3.6) and 15.7 (5. 0)%, respectively. The pattern was similar in idiopathic bladders. CONCLUSION: These observations suggest that the primary defect in the idiopathic and neuropathic bladders is a loss of nerves accompanied by a hypertrophy of the cells. These changes may continue with further hypertrophy of the cells and an increased production of elastin and collagen within the muscle fascicles.     

Role of mast cells and myofibroblasts in human peritoneal adhesion formation

OBJECTIVE: To study fibroblasts and mast cells in human peritoneal adhesions and to evaluate whether their interaction plays a role in adhesion development. SUMMARY BACKGROUND DATA: Myofibroblasts play a critical role in wound repair/fibrosis. Mast cells influence the formation of peritoneal adhesions in a rat model, and they are modulators of fibroblast functions. METHODS: Peritoneal adhesion biopsies were processed for either histology (H&E, toluidine blue) or immunohistochemistry (tryptase, laminin, collagen type IV and VIII, and alpha-SMA) or grown as explants for obtention of fibroblasts. The effects of mast cell (HMC-1) sonicate and selected mast cell mediators and cytokines on fibroblast proliferation ([ (3)H]thymidine) and collagen synthesis ([ (3)H]proline) and on fibroblast contractile activity (tridimensional collagen lattice) were evaluated. Mast cell mediators influencing fibroblast proliferation were partially characterized by enzymatic susceptibility and FPLC gel filtration column chromatography. RESULTS: Most of the fibroblasts in peritoneal adhesions were identified as alpha-SMA-positive myofibroblasts. Mast cell hyperplasia was observed and more than one third of the mast cells were degranulated. Few mast cells showed a faint staining for laminin or collagen type IV and VIII. Mast cell sonicate increased fibroblast proliferation and contractile activity while decreasing collagen synthesis. Mast cell sonicate proliferating activities were found to be proteinase-sensitive with a molecular weight of more than 158 kd, of approximately 40 kd, and of less than 10 kd. TGF-beta and tryptase enhanced collagen synthesis; TNF-alpha and chymase decreased it. None of the selected mediators increased fibroblast proliferation. CONCLUSIONS: Myofibroblasts are the main connective tissue cells present in human peritoneal adhesions, and mast cells play a direct role in peritoneal adhesion formation.     

Morphological and morphometric studies of the human obstructed, trabeculated urinary bladder

As part of an ongoing study on trabeculation of the human urinary bladder, morphological and morphometric techniques have been employed on biopsy samples of detrusor muscle removed from control and urodynamically obstructed patients. In control material the mean profile area, profile diameter and nucleated profile percentage of bladder smooth muscle cells were determined. The values of the same parameters were obtained for smooth muscle cells in samples from urodynamically obstructed and endoscopically trabeculated patients. Comparison of the results obtained from the two groups showed that smooth muscle cells undergo compensatory hypertrophy in response to outflow obstruction. Furthermore, connective tissue infiltration of detrusor muscle bundles is a characteristic of those bladders which possess cells showing the largest increase in cell size.     

神经原性膀胱逼尿肌超微结构的变化

目的　了解低反射神经原性膀胱患者逼尿肌超微结构的改变。　方法　膀胱顶部逼尿肌分别取自 11例神经原性膀胱手术患者和 7例意外死亡新鲜尸体膀胱 ,透射电镜观察两组逼尿肌超微结构差异。　结果　低反射神经原性膀胱逼尿肌有去极化现象 ,超微结构改变包括肌细胞外形不一致、分布不均匀 ,走行紊乱 ;细胞中间连接少 ,常填充以胶原纤维和无定形成分 ;肌质膜下胞饮小泡减少 ;细胞内线粒体减少 ,肌丝走行紊乱 ,致密体分布不均等。正常平滑肌细胞外形、大小基本相似 ,分布均匀 ,走行整齐一致。细胞中间连接多 ,细胞间距小、均匀 ;胞饮小泡与密区沿肌质膜均匀分布 ;细胞内见各种细胞器 ,肌丝及致密体平行整齐排列。　结论　低反射神经原性膀胱逼尿肌超微结构改变可能和逼尿肌去神经性无力 ,逼尿肌尿道括约肌原有平衡失调 ,造成膀胱出口相对阻力增加等有关。神经原性膀胱患者临床上宜及早采取预防措施 ,防止继发性逼尿肌超微结构改变对其收缩能力的进一步影响     

Detrusor collagen content in the denervated rat urinary bladder

Neurogenic bladder dysfunction was created in male rats by removal of the pelvic ganglia. The bladders were then emptied manually once daily, and kept free from infection. After 10 days or six weeks the bladders were taken out and the mucosa-submucosa was removed. The detrusors were then weighed and used for collagen assay. Detrusor weight increased 4.5 and six times after 10 days and six weeks, respectively. Total detrusor collagen increased 2.3 and 3.7 times, but due to the increase in detrusor weight the concentration decreased to 60 per cent of normal. The electron microscopic investigation showed that the cross-sectional area of the smooth muscle cells had increased fourfold after six weeks. The collagen fibrils were found mainly in the interstitial tissue between the muscle bundles. As these increased in size following the neurogenic lesion, the collagen-rich tissue component decreased relatively. Our conclusion is that frequent emptying and the avoidance of bladder infection protects the denervated rat urinary bladder wall from injuries that would otherwise lead to an increased collagen concentration.     

Correlation between mast cell density and myocardial fibrosis in congestive heart failure patients

Besides the well-established role of mast cells in allergic reactions as an important source of vasoactive and proinflammatory products, they have been related to tissue fibrosis and remodelling processes. In a heart failure (HF) animal model, mast cells were shown to synthesize transforming growth factor beta1 and basic fibroblast growth factor in myocardial tissue and were localized to an area with fibrosis. Our objective was to quantify mast cell density in left ventricles from patients with congestive heart failure who were candidates for transplantation and to analyze whether they showed a correlation with the fibrosis level of the same area. METHODS: We obtained myocardial biopsies from 20 patients with end-stage HF secondary to idiopathic dilated cardiomyopathy (iDCM) undergoing heart transplantation and 15 controls (donors without cardiopathy). Mast cells were detected by immunohistochemistry with a human mast cell chymase antibody and fibrosis levels measured with picrosirius red staining of collagen fibrils with later quantification by morphometry. RESULTS: The patients mean age was 51 +/- 3 years. Fibrosis levels in the myocardial sections from patients with congestive HF was three-fold higher than those in control myocardium (12.41 +/- 1.7% vs 3.98 +/- 0.63%, P < .001). Mast cell density correlated with the collagen fraction and could be fitted to a linear regression curve: collagen fraction = 0.78 + 0.05 mast cell density (n = 33, P < .005, R2 = 0.28). CONCLUSION: The elevated collagen fraction present in failing hearts may be the cause of increased stiffness and loss of elasticity that is detected in patients with end-stage HF. Due to the mast cells capacity to synthesize vasoactive and fibrogenic products and the correlation between their density and fibrosis levels, they probably play a role in the ventricular remodelling in HF.     

Extracellular matrix protein expression during mouse detrusor development

Background/Purpose: Extracellular matrix proteins are implicated in regulating cell proliferation and differentiation. The authors systematically analysed the expression of elastin; collagen types I, III, IV; laminin; and fibronectin during mouse detrusor muscle development, a period during which downregulation of detrusor proliferation and increasing smooth muscle differentiation is known to occur. Methods: Embryonic days 14 (E14) and 18 (E18), and postnatal day 1 (D1) and week 6 (6wk) were examined, a period spanning the inception of the bladder to postnatal maturity. Immunohistochemistry of whole bladders was used to immunolocalise protein expression, and Western blot of dissected detrusor layers was used to semiquantify soluble protein expression. Results: All proteins were detected at all 4 stages. Statistically significant increases were documented for elastin (E14 to 6wk), collagen type I (E18 to 6wk), collagen type III (D1 to 6wk) and laminin (E14 to 6wk). Fibronectin levels were relatively high up to D1, after which levels declined significantly. Collagen type IV levels decreased significantly (E18 to 6wk). Conclusions: The authors postulate that changing levels of laminin and fibronectin have opposing effects on the transition from proliferating primitive mesenchymal cells to differentiated detrusor muscle. Furthermore, changes in collagen type III and elastin may be important for bladder compliance. J Pediatr Surg 38:1-12.     

Sequential morphological changes in the pig detrusor in response to chronic partial urethral obstruction

The sequential morphological changes which occur in the wall of the porcine urinary bladder in response to experimentally induced partial outflow obstruction have been determined using morphometric light and electron microscope techniques. The initial morphological response to urethral obstruction was a reduction of approximately 50% in the density of autonomic nerves in the bladder wall which occurred within the first 3 months. During this period the morphology of smooth muscle cells and the distribution of connective tissue were unaltered. Longer periods of obstruction produced a gradual further reduction in the density of innervation. However, from 3 months onwards the detrusor muscle bundles became infiltrated by connective tissue, although significant change in muscle cell size was not detected after obstruction for 12 months. Using electron microscopy, smooth muscle cells were observed to possess extensive amounts of perinuclear granular reticulum, this feature being particularly marked in bladders subjected to longer periods of obstruction. The underlying mechanisms responsible for these structural changes remain to be determined.     

Effects of partial bladder outlet obstruction and its relief on types I and III collagen and detrusor contractility in the rat

Bladder outlet obstruction induces a rapid hypertrophy characterized by increased bladder mass and collagen deposition. An increase in collagen is likely to reduce the contractility and compliance of bladder wall. This study was undertaken to investigate the effects of partial bladder outlet obstruction and its relief on types I and III collagen, and the relationship between detrusor contractility and collagen types. A total of 40 female rats was used for experiment and divided into one control, one obstruction, and three recovery groups. The contractility to field stimulation was recorded; total collagen and collagen concentration were quantified. The localization of types I and III collagen and the expression of pro-alpha1(I) and alpha1(III) collagen mRNA were determined by immunohistochemical staining and Northern blot hybridization, respectively. Contractile response to field stimulation was reduced after obstruction and recovered following relief. The total amount of collagen increased after obstruction and decreased after relief; however, collagen concentration decreased after obstruction and increased following relief. Contractility correlated negatively with total collagen but positively with collagen concentration. The protein deposition of types I and III collagen was localized in lamina propria and muscle bundles in all groups. The expression of types I and III collagen gene was up regulated after obstruction, but down regulated after relief. Negative correlation between contractility and gene expressions of collagen types was significant. These data suggest that the change in localization and quantity of collagen types leads to morphologic changes of bladder and can have an impact on the contractility of detrusor. Neurourol. Urodynam. 19:29-42, 2000.     

Morphological,stereological, and biochemical analysis of the mini-pig urinary bladder after chronic outflow obstruction and after recovery from obstruction

Chronic partial bladder outlet obstruction was created in nine mini-pigs by implanting a 6–7 mm ring around the proximal urethra. After a median obstruction period of 63 days, the ring was removed and after a recovery period of median 60 days the animals were sacrificed. Changes in muscle and connective tissue were assessed by unbiased, modern morphometry and biochemical analysis After obstruction the results were as follows: (1) a 6-fold increase in bladder weight, (2) a 2.5-fold increase in smooth muscle cell size, (3) a 3-fold increase in smooth muscle cell number, (4) unchanged proportions between muscle and connective tissue, (5) unchanged hydroxyproline concentrations, (6) an 8-fold increase in total collagen content, (7) an increase in the ratio of type I/III collagen, and (8) a 7-8-fold increase in total content of type I and III collagen. All changes were markedly, though incompletely, reversed after recovery, except smooth muscle cell number and the ratio of type I/III collagen. © 1995 Wiley-Liss, Inc.     

Altered distribution of interstitial cells in the guinea pig bladder following bladder outlet obstruction

AIMS: We investigated the effects of bladder outlet obstruction (BOO) on the distribution of interstitial cells (ICs) in the guinea-pig bladder. METHODS: Bladder overactivity of BOO animals was validated with urodynamic studies. Immunohistochemical analyses for Kit and vimentin as markers for ICs were performed on both BOO and control bladders. Morphological and functional properties of detrusor smooth muscle (DSM) were examined with alpha-smooth muscle actin staining and intracellular recording, respectively. Electron microscopy was also carried out to characterize ultrastructural morphology of ICs. RESULTS: Two weeks after surgery, BOO animals showed an increased voiding frequency and a reduced voiding volume. Filling cystometry demonstrated a frequent incidence of non-voiding contractions, a reduced interval between voiding contractions and an increased voiding pressure in BOO bladders. In BOO bladders, the thickness of suburothelial and subserosal connective tissue layers was increased, whilst that of detrusor smooth muscle (DSM) layer was less affected. Population of Kit or vimentin immunoreactive ICs was increased in subserosal layers, and their distribution was altered in suburotherial layer in BOO bladders. Neither alpha-actin immunoreactivity nor spontaneous electrical activity of DSM was altered in BOO bladders. ICs were characterized by their numerous mitochondria and caveolae, and had a close contact with each other and with neighboring DSM or nerves. CONCLUSIONS: These results demonstrated the increased population of ICs in the BOO guinea-pig model for the first time, and suggest that the altered distribution of ICs may contribute to the pathophysiology of bladder overactivity.     

Tumor necrosis factor promotes differential trafficking of bladder mast cells in neurogenic cystitis

PURPOSE: IC is often considered neurogenic cystitis, in which mast cells are involved in a positive feedback loop that results in sustained urothelial inflammation. To characterize these processes we developed a murine model of neurogenic cystitis using Bartha's strain of PRV based on a similar model in the rat. MATERIALS AND METHODS: Female C57BL/6 mice (National Cancer Institute, Bethesda, Maryland) were used in the study. Neurogenic cystitis was induced by the injection of Bartha's strain of PRV (2.2 x 10 pfu) into the abductor caudalis dorsalis tail base muscle. Bladder inflammation was assessed by leukocyte influx and Evans blue dye extravasation. Mast cells were visualized in bladder tissue by staining with 0.1% toluidine blue. RESULTS: Inoculation with PRV in the abductor caudalis dorsalis resulted in cystitis within 3 days. Coincident with the induction of cystitis mast cells accumulated in the lamina propria due to mast cell trafficking from the proximal detrusor (relative to the lumen), whereas mast cells from the distal detrusor were unchanged and total mast cell counts were not increased. Degranulated mast cells increased approximately 20-fold in the lamina propria of infected mice relative to controls. In TNF receptor 1/2 deficient mice (Jackson Laboratory, Bar Harbor, Maine) mast cell trafficking was not observed in response to PRV and mast cells were not degranulated. CONCLUSIONS: These data indicate that neurogenic cystitis is associated with the differential trafficking and activation of distinct mast cell pools in the bladder. Since TNF mediates these events, anti-TNF therapy may mitigate the pathogenesis of neurogenic cystitis.     

Increased mast cells of the bladder in suspected cases of interstitial cystitis: a possible disease marker

Mast cells reportedly have been increased in the detrusor muscle bundles of the bladder in patients with interstitial cystitis. In 30 patients with suspected interstitial cystitis quantification of mast cells within the muscularis and submucosa was done. The results were compared to those obtained from a variety of normal bladder specimens removed surgically (16 specimens) and at autopsy (15), and from bladders with a variety of miscellaneous inflammatory conditions (20). In patients with suspected interstitial cystitis the number of mast cells was increased significantly (p less than 0.001) compared to the 3 control groups. This finding suggests that quantification of mast cells in the muscularis may be a useful marker in the histopathological evaluation of bladder biopsies in patients with suspected interstitial cystitis.     

Effects of imatinib mesylate (Glivec) as a c-kit tyrosine kinase inhibitor in the guinea-pig urinary bladder

AIMS: In the gastrointestinal tract, slow wave activity in smooth muscle is generated by the interstitial cells of Cajal (ICC). Detrusor smooth muscle strips of most species show spontaneous contractions which are triggered by action potential bursts, however, the pacemaker mechanisms for the detrusor are still unknown. Recently, ICC-like cells have been found in guinea-pig bladder, using antibodies to the c-kit receptor. We have investigated the effects of Glivec, a c-kit tyrosine kinase inhibitor, on spontaneous action potentials in guinea-pig detrusor and intravesical pressure of isolated guinea-pig bladders. METHODS: Changes in the membrane potential were measured in guinea-pig detrusor smooth muscle using conventional microelectrode techniques. Pressure changes in the bladder were recorded using whole organ bath techniques. RESULTS: Smooth muscle cells in detrusor muscle bundles exhibited spontaneous action potentials, and spontaneous pressure rises occurred in isolated bladders. Glivec (10 microM) converted action potential bursts into continuous firing with no effects on the shape of individual action potentials. Glivec (>50 microM) reduced the amplitude of spontaneous pressure rises in the whole bladder in a dose dependent manner and abolished spontaneous action potentials in detrusor smooth muscle cells. CONCLUSIONS: The results suggest that ICC-like cells may be responsible for generating bursts of action potentials and contractions in detrusor smooth muscle. Drugs inhibiting the c-kit receptor may prove useful for treating the overactive bladder.     

Presence and passage dependent loss of biochemical M3 muscarinic receptor function in human detrusor cultured smooth muscle cells

PURPOSE: We explored morphological and biochemical aspects of human detrusor cells in culture as a tool for investigating the physiological mechanisms underlying bladder function and disturbances. MATERIALS AND METHODS: Primary cultures of smooth muscle cells were derived from human bladder specimens of patients with an average age of 70 years undergoing cystectomy. Cultured cells were investigated by morphological, immunocytochemical and Western blot analysis. The alpha-actin content as well as the presence of muscarinic M2 and M3 receptors was determined in cell lysates and in fresh tissue homogenate for comparison. The functional response to muscarinic stimulation was assessed by measuring IP3 production induced by 1 mM. carbachol. RESULTS: Cultured smooth muscle cells showed a characteristic spindle-shaped morphology at early passages. Similarly immunocytochemical and Western blot analysis revealed an alpha-actin cell content that was unmodified up to passage 3. Conversely this marker protein sharply decreased during further passages. M3 muscarinic receptor was present in cultured cells and fresh tissue homogenates, whereas the M2 subtype was evident only in homogenates. Carbachol produced a time dependent increase in IP3 cell content, reaching maximal production after 20 minutes of exposure. This response was passage sensitive. CONCLUSIONS: Cultured human detrusor smooth muscle cells maintain their morphological and biochemical characteristics up to passage 3. With this caveat such cells can be an appropriate tool for investigating the molecular pathways underlying cholinergic activation in normal physiological and pathological bladders, and accordingly for detecting putative targets for pharmacological intervention.